Treatment of disease by modulation of SIRT6

ABSTRACT

An aspect of an embodiment of the invention relates to providing treatment of disease, in particular age-related disease, through increasing or decreasing the activity of SIRT6 protein. This may be accomplished through upregulation and downregulation of expression of SIRT6 in mammals. It has been found by the inventors that mice over-expressing SIRT6 have a longer lifespan in comparison to control mice, indicating that increasing SIRT6 expression can lengthen lifespan of mammals. Agents which modulate SIRT6 expression through, for example binding to 3′UTR region of human mRNA encoding SIRT6 or by blocking binding of agents to 3′UTR region of human mRNA encoding SIRT6, have been identified.

RELATED APPLICATIONS

The present application is a US National Phase of PCT Application No.PCT/IB2013/050607, filed on Jan. 24, 2013, which claims the benefitunder 35 U.S.C. 119(e) of U.S. Provisional Application 61/632,391 filedon Jan. 24, 2012, the disclosure of which are incorporated herein byreference.

TECHNICAL FIELD

Embodiments of the invention relate to treatment of diseases.

BACKGROUND

Sirtuin proteins (also known as Sir2 proteins and silent mating typeinformation regulation 2 proteins) are a family of proteins thatregulate biological pathways in various organisms including bacteria,yeasts and mammals. Yeast sirtuin has been well characterized, andhomologs known as SIRT1 through SIRT7 have been identified in humans.These proteins may act as deacetylases, lysine malonyl transferases orADP (adenosine diphosphate) ribosyl transferases and regulate variousactivities within cells.

The sirtuin-6 (SIRT6) protein, encoded by the SIRT6 gene, is a proteinthat has been associated with energy metabolism.

SUMMARY

An aspect of an embodiment of the invention relates to providingtreatment of disease through increasing or decreasing the activity ofSIRT6 protein. This may be accomplished through upregulation anddownregulation of expression of SIRT6 in mammals. The effect ofup-regulating and down-regulating SIRT6 expression was shown in animalmodels. Although some mammalian sirtuins were previously shown toregulate age-related diseases, mice overexpres sing SIRT1 retain thesame lifespan as control wild type (WT) mice. It has been found by theinventors that mice over-expressing SIRT6 have a longer lifespan incomparison to control mice, indicating that increasing SIRT6 expressioncan lengthen lifespan of mammals.

According to an aspect of an embodiment of the invention therapeuticagents which increase the activity of SIRT6 or upregulate SIRT6expression may be used to increase human lifespan and to control agerelated diseases. “Increasing activity of SIRT6” may refer to bothincreasing protein activity, quality or quantity in an organism and toupregulating expression of the SIRT6 gene, thereby producing SIRT6protein to a greater extent.

An aspect of an embodiment of the invention relates to providing amethod for treating an age-related disease comprising administering to ahuman patient in need thereof an agent which increases the expression ofthe SIRT6 gene.

An embodiment of the invention relates to providing a method fortreating an age-related disease comprising administering to a humanpatient in need thereof an agent which activates SIRT6 protein in ahuman patient.

An embodiment of the invention relates to providing a method fortreating cancer comprising administering to a human patient in needthereof an agent, to decrease the activity of SIRT6 in cancer cells.“Decreasing activity of SIRT6,” may refer to both decreasing proteinactivity, quality or quantity in an organism and to downregulatingexpression of the SIRT6 gene, thereby producing SIRT6 protein to alesser extent.

In the discussion unless otherwise stated, adjectives such as“substantially” and “about” modifying a condition or relationshipcharacteristic of a feature or features of an embodiment of theinvention, are understood to mean that the condition or characteristicis defined to within tolerances that are acceptable for operation of theembodiment for an application for which it is intended.

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the detaileddescription. This summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF FIGURES

Non-limiting examples of embodiments of the invention are describedbelow with reference to figures attached hereto that are listedfollowing this paragraph.

FIGS. 1a and 1b depict survival curves showing percent survival overtime for transgenic SIRT-6 overexpressing (SIRT6-tg) male (FIG. 1a ) andfemale (FIG. 1b ) mice from a strain designated as line 55 and WTcontrol mice;

FIGS. 1c and 1d depict survival curves showing percent survival overtime for SIRT6-tg male (FIG. 1c ) and female (FIG. 1d ) mice from astrain designated as line 108 and WT control mice;

FIGS. 1e and 1f depict graphs showing results of a glucose tolerancetest in SIRT6-tg male (FIG. 1e ) and female (FIG. 1f ) mice and WTcontrol mice (old mice, aged 19 months) alongside bar graphs depictingarea under curve (AUC) for each group;

FIG. 1g depicts a graph showing percentage body fat in mice aged twoyears from SIRT6-tg and WT control mice groups;

FIG. 1h depicts a graph showing body weight, in grams in mice aged twoyears from SIRT6-tg and WT control mice groups;

FIG. 2 depicts a human DNA sequence (referred to as SEQ. ID NO. 1)corresponding to mRNA encoding SIRT6, including its three primeuntranslated (3′UTR) region;

FIG. 3 depicts the 3′UTR region of human mRNA encoding SIRT6 (referredto as SEQ. ID NO. 2) with nucleotides 479-486 and nucleotides 386-394underlined, and nucleotides 199-206, nucleotides 350-359 and nucleotides455-463 double underlined;

FIG. 4 depicts miRNA sequences of miR-33a, miR-33b, miR-122, miR-370referred to as SEQ. ID NO. 3, SEQ. ID NO. 4, SEQ. ID NO. 5 and SEQ. IDNO. 6 respectively, agents which may be used to downregulate SIRT6expression in accordance with an embodiment of the invention;

FIG. 5 depicts miRNA sequences of miR-33a, miR-33b and miR-122, referredto as SEQ. ID NO. 3, SEQ. ID NO. 4 and SEQ. ID NO. 5, respectively boundto their corresponding binding sites on 3′ UTR SIRT6 mRNA;

FIG. 6 depicts sequences of antimiR-33a, antimiR-33b and antimiR-122,referred to as SEQ. ID NO. 7, SEQ. ID NO. 8, SEQ. ID NO. 9 and SEQ. IDNO. 10, respectively, agents which may be used to upregulate SIRT6expression in accordance with an embodiment of the invention;

FIG. 7A-B show downregulation of SIRT6 expression in cells transfectedby miR-33a (FIG. 7A), and miR-122 (FIG. 7B), as indicated using westernblot analysis by lighter band in miR-transfected cells as compared tocontrol (indicated by “−”) and vehicle cells;

FIGS. 8A-B show upregulation of SIRT6 expression in cells transfected byantimiR-33a (FIG. 8A) and antimiR-122 (FIG. 8B) as indicated by westernblot analysis by heavier band in antimiR-transfected cells as comparedto control and scramble sequence (sc) transfected cells;

FIG. 9 shows upregulation SIRT6 expression in cells transfected byantimiR-370 and downregulation of SIRT6 expression in cells tranfectedby miR-370 as indicated by western blot analysis by a heavier band inantimiR-transfected cells as compared to WT cells and by a lighter bandin miR tranfected cells, as compared to WT cells.

DETAILED DESCRIPTION

The examples detailed in the following paragraphs illustrate thetherapeutic effect of upregulation or downregulation of SIRT6. Inparticular, example 1 illustrates that SIRT6 upregulation in mammalsprolongs lifespan and may prevent age related disease. Example 2describes agents that have been identified to upregulate anddownregulate SIRT6 expression and may be used, according to embodimentsof the invention, for treatment of age-related disease.

Example 1A Increased Lifespan of Transgenic Mice Over-Expressing SIRT6

In this example, lifespan of Sirt6-tg mice was examined in comparison totheir control littermates. Sirt6-tg mice were produced using CB6F1strain, and maintained on a segregating stock containing equalcontributions of C57BL/6J and BALB/cOlaHsd (Harlan laboratories)backgrounds, both considered to be long lived strains, as previouslydescribed (Yuan 2009). The experiment was performed on 245 mice (119males and 126 females) from two transgenic lines generated from twoseparate founders.

In a period of about three months, each male founder (heterozygous forthe transgene) was bred with WT CB6F1 females and the progeny were usedfor the life-span analysis. The integration site in each line of thetransgene was mapped by nested inverse polymerase chain reaction(“PCR”), with several different restriction enzymes from both the rightand left of the integration site and found to be in noncoding regions.Results were validated with sequencing and additional PCR confirmingintegration sites. Mice were kept under specific pathogen freeconditions in individually ventilated cages, “IVC cages” that wereroutinely examined and found negative for viral serology and both endoand ecto-parasites. The mice were raised under 12 h day/night conditionsand had free access to standard chow diet and water. All mice were leftundisturbed until natural death. Mice tested at all ages were not partof the life-span cohort.

Log-rank test analysis showed significant differences in the survivalcurves between WT and transgenic male mice lines 55 and 108 (FIGS. 1aand 1c respectively) but not between WT and transgenic female mice inboth lines (FIGS. 1b and 1d .) In male Sirt6-tg mice median lifespanincreased by 14.5% and 9.9% and mean lifespan increased by 14.8% and16.9% relative to male WT littermates, lines 55 and 108 respectively. Infemale Sirt6-tg mice no significant increase in median or mean lifespanwas found relative to female WT littermates in both lines. Males'maximum lifespan (mean lifespan of oldest 10% within a cohort to die)increased by 15.8% and 13.1% relative to that of WT littermates mice,lines 55 and 108 respectively. In summary, SIRT6 overexpressionincreased longevity in males but not in females.

SIRT6 was shown to regulate genome stability and metabolism, two majorcontributors to longevity. Loss of genomic stability is known to be animportant aspect of cancer. Postmortem gross and microscopic examinationof mice revealed malignant tumors in a variety of organs, although thehighest incidence of tumors in all mice was found to be in the lungs. Acomparison of median lifespan between WT and Sirt6-tg mice with lungtumors shows a trend of increased lifespan in transgenic mice of 11.7%.Therefore, it is suggested that SIRT6 has an effect on cancer, inparticular, in lung cancer which plays a role in SIRT6's pro-longevityeffect.

SIRT6 might positively affect age-associated metabolic disorders such asdeclining insulin sensitivity and impaired glucose tolerance. Glucosemetabolism was tested using the glucose tolerance test (GTT.) Mice weredeprived of food overnight prior to GTT. 2 g (gram) of glucose per kg(kilogram) of body weight was injected via intraperitoneal injection.Blood glucose was measured on samples obtained by tail bleeding prior toglucose administration and after 15, 30, 60, 90, and 120 min (minutes)using glucometer test strips (Abbott).

No significant differences in glucose metabolism were found in youngmice (4-7 months old). However, GTT showed that aged SIRT6-tg mice (19months old, the maximal age of WT mice before appearance of major death)display a trend towards improved glucose homeostasis as evident in FIG.1e (males, 6 mice per genotype) and 1 f (females, 4 mice per genotype).Area under curve (AUC) for each GTT is shown on right. Values areexpressed as mean±SEM. Asterisk signifies P<0.05, two-tailed t-test.

Statistical analyses were performed in SPSS (Version 17, SPSS Inc.)software using unpaired t tests, ANOVA, log rank and Cox regressionanalysis. Quantile regression was performed in order to analyze maximumlife-span according to Wang et al. 2004, and P values were calculatedusing Fisher's exact test. Each line and gender were analyzed forlife-span parameters separately. Results were expressed as mean±SEM.P<0.05 was regarded as statistically significant.

Example 1B Decreased Body Fat of 2 Year Old Mice in Transgenic MiceOver-Expressing SIRT6

Transgenic mice were prepared as in example 1A. Body weight wasdetermined in male and female transgenic and wild type mice at two yearsof age. In addition, percentage of body fat was determined using NMRtechnology.

As shown in FIG. 1G, percentage of body fat for transgenic males wassignificantly lower than percentage of body fat for wild type males. Asshown in FIG. 1H, weight was significantly lower for transgenic malesthan for wild type males. No significant differences in these twomeasures were found between wild type and transgenic females.

These results indicate that upregulation of SIRT6 may preventage-induced obesity and diseases associated with age-induced obesity.Although the diets of both transgenic and wildtype groups wereidentical, and were not high-fat diets, the transgenic overexpressingmice showed less age-related weight gain and less age-related increasedbody fat content.

Example 2 RNA Interference

In mammalian cells, genetic material is stored in DNA in the cells'nuclei. In order to convert a cell's DNA into a protein or enzyme,transcription occurs in the cell nucleus, in which the DNA is used as atemplate which is “copied” to messenger RNA (mRNA) which is thentransported out of the nucleus of the cell to be translated into aprotein or enzyme at a ribosome, in the cell's cytoplasm. In thetranslation process, a ribosome builds a protein or enzyme based on thesequence of the mRNA.

The translation process is moderated in cells in many organisms in aprocess called RNA interference (RNAi.) In RNAi, small pieces of RNAcalled microRNA (miRNA) or small interfering RNA (siRNA) specificallyblock the translation of genes into proteins. RNAi technology can beused by introducing synthetic siRNA or miRNA into cells, therebyblocking expression of specific genes.

MiRNA is a short strand of RNA (about 20 nucleotides) which binds to aportion of mRNA and prevents the mRNA translation, thereby effectively“silencing” a gene. Generally, miRNA binds to a non-coding region of themRNA, the three prime untranslated (3′UTR) region, but may also bindother regions. There are about 1000 naturally occurring miRNA sequencesthat have been identified to date.

The inventors have determined that the sequences of 4 human miRNAs arecomplementary to portions of the 3′UTR region of human SIRT6 mRNA. The 4miRNAs which have been identified are miR-33a, miR-33b, miR-122 andmiR-370. Their respective sequences SEQ. ID NO. 3, SEQ. ID NO. 4, SEQ.ID NO. 5 and SEQ. ID NO. 6 are depicted in FIG. 4, and binding sites ofSEQ. ID NO. 3-5 on 3′UTR SIRT6 mRNA are shown in FIGS. 3 and 5. In FIG.3, the binding sites of miR-122 and miR-33 are underlined, and thepresumed binding sites of miR-370, nucleotides 199-206, nucleotides350-359 and nucleotides 455-463, are double-underlined. The boldcharacters in FIG. 5 represent the presumed nucleotides of the miRNAinvolved in binding.

MiR-33a, miR-33b, miR-122 and miR-370 were cloned according to thefollowing procedure. The human hepatoma cell line, known as HepG2, wasused and grown in full DMEM medium supplemented with 10% fetal calfserum, penicillin-streptomycin mixture 100 units/ml and L-glutamine 100units/ml (units/milliliters). For transfection, cells were grown to 50%confluence in a 6-well cell culture plate. After 24 hours, transfectionwas performed using the transfection reagent lipofectamine (Invitrogen.)The genomic DNA that encompass the miR in addition to 200 base pairsupstream and downstream was cloned and insterted into a vector calledMirvec. The following amounts of DNA were transfected: SIRT6: 2 μg(microgram) in a 6-well format. The total amount of DNA in eachtransfection was normalized with apcDNA3.1+vector. The cells wereharvested 48 hours post transfection.

48 hours after transfection, western blot was performed using thefollowing procedure: HepG2 cells were detached by scraping and thenresuspended in lysis buffer [50 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.5), 150mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM ethylenediaminetetraaceticacid, 1 mM ethylene glycol tetraacetic acid, and a protease inhibitorcocktail (diluted at 1:1000)], and incubated on ice for 15 min. Lysateswere cleared by centrifugation (13,000 rpm, 15 minutes). For directelectrophoretic analysis, sample buffer (6×) was added to cell lysates,that were subsequently resolved by SDS-polyacrylamide gel andelectrophoretically transferred to a nitrocellulose membrane. Membraneswere blocked in Tris-Buffered Saline and Tween 20 (TBST) buffer [0.02MTris-HCl (pH 7.5), 0.15M NaCl, and 0.05% Tween 20] containing 5% BSA,blotted with a primary antibody for 1 hour or overnight, washed in TBST,and incubated for 30 min with a secondary antibody linked to horseradishperoxidase. Immunoreactive bands were detected using enhancedchemiluminescence (ECL) reagents (Pierce, Rockford, Ill. USA).

FIG. 7 shows results of a western blot analysis of SIRT6 expression inmiR transfected cells as compared to control, untreated cells (−) and avehicle vector. The actin bands serve as a loading control. Densitometerquantification of SIRT6 was performed for each band and was expressedrelative to control, non-treated cells. These results show thattransfection of miR-33a and miR-122 decreased the expression of SIRT6 intransfected cells.

An antimiR (also known as an antagomir) is a small synthetic RNA that iscomplementary to a specific miRNA target, and inhibit miRNA. AntimiRtechnology has been successfully used to inhibit fibrosis of heart andlung. AntimiR-33a, antimiR-33b, antimiR-122 and antimiR-370(complementary to miR-33a, miR-33b, miR-122 and miR-370 respectively)were obtained from Applied Biosystems. The sequences are shown in SEQ.ID NO. 7, SEQ. ID NO. 8, SEQ. ID NO. 9 and SEQ. ID NO. 10 respectively,in FIG. 6.

The miRNA was transfected into HepG2 cells via reverse transfection.Cells were seeded in a 6-well plate. 24 hours after seeding they weretransfected with antimiR-33a, antimiR-33b, antimiR-122 or antimiR-370(final concentration: 30 nM).

In order to prepare the transfection medium, in a first tube, 100 μl(microliter) of serum-free medium was added to 5 μl siPORT NeoFX, alipid based transfection agent available from Applied Biosystems, andthe tube was mixed gently. In a second tube, 100 μl of serum-free mediumwas added to 7.5 μl antimiR (10 μM) and was mixed gently. After 10 min,the first and second tubes were mixed gently, and then after 10 min, themixture was dispensed into an empty well, and 500,000 cells were seededin each well to a final well volume of 2.5 ml. The wells were tiltedback and forth. The cells were harvested 40-48 hours post transfectionfor further analysis in western blot.

Western blot was performed as described above. FIG. 8 shows results ofSIRT6 expression using a western blot analysis of anti-miR transfectedcells as compared to control, untreated cells (−) and cells transfectedwith a scramble sequence (sc). The actin bands serve as a loadingcontrol. Densitometer quantification of SIRT6 was performed for eachband and was expressed relative to control, non-treated cells.Transfection of antimiR-33a and antimiR-122 increased the expression ofSIRT6 in transfected cells.

FIG. 9 shows results of SIRT6 expression using a western blot analysisof antimiR-370 and miR-370 transfected cells as compared to control,untreated cells (wt). The actin bands serve as a loading control. Theseresults show that transfection of miR-370 decreased the expression ofSIRT6 in transfected cells, and transfection of anti-miR-370 increasedthe expression of SIRT6 in transfected cells.

AntimiR-33a, AntimiR-33b, AntimiR-122 and AntimiR-370 may each be agentsuseful for increasing expression of SIRT6 when administered to patientsin need thereof.

Example 3 Identification of Agents Capable of Modulating SIRT6 Activity

In addition to the methods described above, a fluorometric assay wasperformed to identify agents capable of modulating SIRT6, includingagents capable of activating SIRT6 using the following method.

The assay employs a short acetylated peptide based on amino acids 5-9 ofhistone H3K9 comprising an acetylated lysine residue, and labeled with afluorophore, 7-amino-4-methylcoumarin (AMC) conjugated to the C-terminalend of the peptide. The AMC fluorophore emits fluorescence when free,but does not emit significant fluorescence in the conjugated state.

The procedure comprises two steps, both performed in the samemicroplate. In the first step, the substrate is incubated with humanrecombinant SIRT6 protein along with the co-enzyme Nicotinamide adeninedinucleotide (NAD+). SIRT6 will act to deacetylate the peptide. Upondeacetylation, the liberated ε-amino group of the lysine becomes atrypsin substrate. Treatment with trypsin in the second step releasesthe fluorophore resulting in an increase in fluorescence (excitation andemission at 350-360 nm and 450-465 nm, respectively). In the first step,SIRT6 can be incubated with agents capable of increasing or decreasingSIRT6 deacetylation activity. Increase in SIRT6 deacetylation may bedetected by increased fluorescence relative to control microplates inwhich no modulating agent is added, and decrease in SIRT6 deacetylationmay be detected by decreased fluorescence relative to controlmicroplates.

Agents identified by this method which increase SIRT6 activity may beeffective in treating age-related disorders in humans. Agents identifiedby this method which decrease SIRT6 activity may be effective intreating cancer in humans.

Example 4 Treatment of Mammals, Including Humans, Using Agents thatModulate SIRT6 Expression

As shown in examples 1A and 1B, upregulation of SIRT6 expression inmammals may increase life span and reduce age-related obesity. Inaddition, upregulation of SIRT6 expression in mammals has shown effectin diseases relating to glucose metabolism and cancer. Accordingly, anaspect of an embodiment of the invention relates to providing a methodfor treating an age-related disease comprising administering to a humanpatient in need thereof an agent which increases the expression of theSIRT6 gene.

As shown in example 2, agents were found which can bind to the mRNAsequence of SIRT6 (SEQ. ID NO. 2) and downregulate its expression(decrease SIRT6 expression) and therefore may be used to treat disease.In addition, it has been found that there are agents which upregulateexpression of SIRT6 (increase SIRT6 expression) by interfering withnaturally occurring agents that downregulate SIRT6 expression, andtherefore may be used to treat disease, in particular age-relateddisease.

In an embodiment of the invention, the age-related disease is selectedfrom the group consisting of neurodegenerative disease, cancer,cardiovascular disease, obesity, type 2 diabetes, increased cholesterollevels, hypertension, ocular disorders including cataracts and glaucoma,osteoporosis, blood clotting disorders, arthritis, hearing loss, strokeand Alzheimer's disease. In an embodiment, the age-related disease istreated in a patient above the age of 50. In an embodiment, theage-related disease is treated in a patient above the age of 65. In anembodiment, the age-related disease is treated in a patient above theage of 80.

In an embdodiment of the invention, the neurodegenerative disease isassociated with degeneration of neural tissue. In an embdodiment of theinvention, the neurodegenerative disease is selected from the groupconsisting of: Alzheimer's disease, Parkinson's disease, Huntington'sdiseases, amyotrophic lateral sclerosis and multiple system atrophy.

In an embodiment of the invention, cardiovascular disease is a diseaseafflicting the heart and/or blood vessels. In an embodiment of theinvention, cardiovascular disease is selected from the group consistingof: coronary heart diseases, cardiomyopathy, hypertensive heartdiseases, cardiac dysrhythmias, endocarditis, cardiomegaly, myocarditis,calcular heart diseases, cerebrovascular disease and peripheral arterialdisease.

In the treatment of age related diseases, agents that increase theactivity of SIRT6 may be administered systemically, for exampleintravenously. In an embodiment of the invention, an agent capable ofmodulating activity of SIRT6, hereinafter, “active agent,” is combinedwith at least one pharmaceutically acceptable excipient to form apharmaceutical composition comprising an active agent. In an embodimentof the invention, the pharmaceutical composition is adapted for human oranimal use via oral, rectal, vaginal, topical, nasal, ophthalmic,transdermal, subcutaneous, intramuscular, intraperitoneal or intravenousadministration.

The pharmaceutical compositions according to an embodiment of theinvention may be conveniently presented in unit dosage form and areprepared by any of the methods well known in the art of pharmacy. In anembodiment of the invention, the unit dosage form is in the form of atablet, capsule, lozenge, wafer, patch, ampoule, vial or pre-filledsyringe.

The pharmaceutical compositions according to the present invention aregenerally administered in the form of a pharmaceutical compositioncomprising at least one active component together with apharmaceutically acceptable carrier or diluent.

For oral administration a pharmaceutical composition can take the formof solutions, suspensions, tablets, pills, capsules, powders, and thelike. Tablets containing various excipients such as sodium citrate,calcium carbonate and calcium phosphate are employed along with variousdisintegrants such as starch and preferably potato or tapioca starch andcertain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type are also employed as fillers in soft and hard-filledgelatin capsules; preferred materials in this connection also includelactose or milk sugar as well as high molecular weight polyethyleneglycols. When aqueous suspensions and/or elixirs are desired for oraladministration, the components of this invention can be combined withvarious sweetening agents, flavoring agents, coloring agents,emulsifying agents and/or suspending agents, as well as such diluents aswater, ethanol, propylene glycol, glycerin and various like combinationsthereof.

The compositions according to embodiments of this invention may also beadministered in a controlled release formulation such as a slow releaseor a fast release formulation. Such controlled release dosagecomposition may be prepared using methods well known to those skilled inthe art.

For purposes of parenteral administration, solutions in sesame or peanutoil or in aqueous propylene glycol can be employed, as well as sterileaqueous solutions of the corresponding water-soluble salts. Such aqueoussolutions may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. These aqueoussolutions are especially suitable for intravenous, intramuscular,subcutaneous and intraperitoneal injection purposes.

Pharmaceutical compositions according to embodiments of the inventionmay contain an amount of 0.1%-95% of the active agent, preferably1%-70%.

In an embodiment of the invention, the daily dosage of the active agentis between 0.001 mg and 3000 mg.

In an embodiment of the invention, the agent which upregulates theexpression of the SIRT6 gene interferes with the activity of miRNA.

Various antimiRs have proven to be effective in mammalian administrationin upregulation of gene expression. AntimiRs have been used in treatmentof hepatitis C virus-infected non-human primates. In an embodiment ofthe invention, the agent is anti-miRNA. In an embodiment, the anti-miRNAis antimiR-33a, antimiR-33b, antimiR-122 or antimiR-370, or acombination thereof.

Administration of antimiRs may be administered to a patient and willtarget specific cells in which there is a high presence of a specificmiRNA. In an embodiment of the invention, miRNA or antimiR isadministered to a patient and modulates SIRT6 expression in adipocytesor hepatocytes.

Downregulation of SIRT6 expression in mammalian cells may cause celldeath in such cells. Downregulation of SIRT6 expression may be targetedto mammalian cancer cells, thereby causing cell death in those cells,thereby treating cancer in the mammal.

In the treatment of tumors, agents that downregulate expression of SIRT6may be injected directly into tumors, thereby causing cell death withincancer cells.

In an embodiment of the invention, the agent which downregulates theexpression of the SIRT6 gene is miRNA. In an embodiment, the miRNA has aregion complementary to a region of SEQ. ID NO. 2 (in FIG. 3). In anembodiment, the miRNA has a region which is complementary to nucleotides479-486 or nucleotides 386-394 of SEQ. ID NO. 2. In an embodiment, themiRNA is miR-33a, miR-33b or miR-122.

In addition to SIRT6 as depicted in SEQ. ID NO. 1 and SEQ. ID NO. 2(FIGS. 2 and 3, respectively), other variants of human SIRT6 exist(including a shorter variant with 27 fewer amino acids,) and methods ofupregulating and downregulating expression of variants of human SIRT6are encompassed according to embodiments of the invention.

In addition to the miRNA and antimiR agents disclosed in example 2,siRNA is another type of RNAi which may be used to modulate expressionof SIRT6 in mammalian cells by upregulating or downregulatingexpression, thereby treating the mammal.

RNA interfering agents can be delivered in a pharmaceutically acceptablecarrier. One or more RNA interfering agents can be used simultaneously.A viral-mediated delivery mechanism can also be employed to deliversiRNAs to cells as described previously (Kim 2007).

There are a number of methods of delivery of siRNA, antimiR and miRNA tomammals in need of RNA interference. Non-selective approaches todelivery include linking cholesterol groups to chemically modifiedsiRNAs for systemic delivery. In addition, siRNAs can be deliveredsystemically by stable nucleic acid-lipid particles (SNALPs).Alternatively, selective approaches can be used, including usingAptamer-siRNA chimaeras to allow siRNAs to be delivered to specific celltypes that display receptors recognized by the aptamers. Heavy-chainantibody fragments (Fabs) and siRNAs can be linked with protamine todeliver siRNAs to specific cell-surface receptors. Nanoparticles thatdisplay specific ligands on their surfaces can be used to target siRNAsto particular cell types. Long-term RNAi may be mediated by viralexpression vectors.

It is suggested according to embodiments of the present invention thatcancer may be treated by upregulation of SIRT6 in an organism. Thiseffect was evident in mice in example 1A. Without being bound by theory,it is suggested that upregulation of SIRT6 may have one or more of thefollowing effects: 1. Increased levels of SIRT6 inhibit phosphorylationof c-Jun N-terminal kinase (JNK), leading to increased heat shockprotein 72 (HSP72) levels, impacting obesity, neurodegenerativedisorders, immune system function and cancer. 2. SIRT6 protects normalcells from DNA damage by stabilizing telomere matter. 3. Cancer cellsare known to reproduce at a higher rate than normal cells based on the“Warburg effect” in which cancer cells rely on glycolysis formetabolism. SIRT6 expression is associated with an anti-glycolyticeffect, thereby preventing cancer cells from increased metabolism andeliminating their ability to survive. 4. SIRT6 overexpression results inactivation of AMP-activated protein kinase (AMPk) due to a significantincrease in total AMPK, phosphorylated AMPK (Thr 172) and AMP/ATP ratio.Metformin, and thiazolidinedione-TZD, known AMPK activators, are to datethe most subscribed medicine used for the treatment of type 2 diabetes.Thus, SIRT6 overexpression will impact diabetes and its associateddisorders. In addition, AMPK is also known to inhibit the Warburgeffect. Thus SIRT6 overexpression may block the aforementioned Warburgeffect via activation of AMPK and may thereby limit cancer progression.

There is further provided, in accordance with an embodiment of theinvention, a method for treating an age-related disease comprisingadministering to a human subject in need thereof an effective amount ofan agent which increases the activity of sirtuin-6 (SIRT6). In anembodiment of the invention, the activity of SIRT6 is increased byadministering to a human subject in need thereof an effective amount ofan agent which increases the expression of the sirtuin-6 (SIRT6) gene.Optionally, the age-related disease is selected from the groupconsisting of: neurodegenerative disease, cancer, cardiovasculardisease, obesity, type 2 diabetes, increased cholesterol levels,hypertension, ocular disorders including cataracts and glaucoma,osteoporosis, blood clotting disorders, arthritis, hearing loss, strokeand Alzheimer's disease. Optionally, the agent increases the expressionof the SIRT6 gene by inhibiting binding of a SIRT6 expression inhibitorto a 3′UTR region of human mRNA encoding SIRT6. Optionally, the SIRT6expression inhibitor binds to a region of SEQ. ID NO. 2. Optionally, theSIRT6 expression inhibitor binds to nucleotides 479-486, nucleotides386-394, nucleotides 199-206, nucleotides 350-359 or nucleotides 455-463of SEQ. ID NO. 2. Optionally, the SIRT6 expression inhibitor comprisesmicro-RNA. Optionally, the SIRT6 expression inhibitor comprises one ormore of SEQ. ID NO. 3, SEQ. ID NO. 4, SEQ. ID NO. 5 and SEQ. ID NO. 6.Optionally, the agent comprises one or a combination of one or more ofSEQ. ID NO. 7, SEQ. ID NO. 8, SEQ. ID NO. 9 and SEQ. ID NO. 10.Optionally, the agent is in the form of a composition in combinationwith a pharmaceutically acceptable carrier. Optionally, the compositionis administered parenterally or optionally intravenously. Optionally,the subject is aged above 50 years old, above 65 years old or above 80years old. Optionally, the daily dosage of agent administered is between0.01 and 3000 mg daily.

There is further provided, in accordance with an embodiment of theinvention, a method for treating cancer comprising administering to ahuman patient in need thereof an agent which decreases SIRT6 activity incancer cells. Optionally, the agent decreases expression of the SIRT6gene. Optionally, the agent comprises a SIRT6 expression inhibitor whichbinds to a 3′UTR region of human mRNA encoding SIRT6. Optionally, theSIRT6 expression inhibitor binds to a region of SEQ. ID NO. 2.Optionally, the agent binds to nucleotides 479-486, nucleotides 386-394,nucleotides 199-206, nucleotides 350-359 or nucleotides 455-463 of SEQ.ID NO. 2. Optionally, the SIRT6 expression inhibitor comprises miRNA.Optionally, the SIRT6 expression inhibitor comprises one or more of SEQ.ID NO. 3, SEQ. ID NO. 4, SEQ. ID NO. 5 and SEQ. ID NO. 6. Optionally,the agent is in the form of a composition in combination with apharmaceutically acceptable carrier. Optionally, the composition isadministered parenterally or intraveneously. Optionally, the dailydosage of agent administered is between 0.01 and 3000 mg daily.

In the description and claims of the present application, each of theverbs, “comprise,” “include” and “have,” and conjugates thereof, areused to indicate that the object or objects of the verb are notnecessarily a complete listing of components, elements or parts of thesubject or subjects of the verb.

Descriptions of embodiments of the invention in the present applicationare provided by way of example and are not intended to limit the scopeof the invention. The described embodiments comprise different features,not all of which are required in all embodiments of the invention. Someembodiments utilize only some of the features or possible combinationsof the features. Variations of embodiments of the invention that aredescribed, and embodiments of the invention comprising differentcombinations of features noted in the described embodiments, will occurto persons of the art. The scope of the invention is limited only by theclaims.

WORKS CITED

-   Kim D H, Rossi J J: Strategies for silencing human disease using RNA    interference. Nat. Rev. Genet. 8, 173-184 (2007).-   Wang, C., Li, Q., Redden, D. T., Weindruch, R. & Allison, D. B.    Statistical methods for testing effects on ‘maximum lifespan’. Mech.    Ageing Dev. 125, 629-632 (2004).-   Yuan et al. Aging in inbred strains of mice: study design and    interim report on median lifespans and circulating IGF1 levels.    Aging Cell. 2009.

The invention claimed is:
 1. A method for treating an age-relateddisease comprising administering to a human subject above the age of 50in need thereof an effective amount of an agent which increases theactivity of sirtuin-6 (SIRT6) by increasing the expression of the SIRT6gene, wherein the agent inhibits binding of a SIRT6 expression inhibitorto a 3′UTR region of human mRNA encoding SIRT6, wherein the expressioninhibitor binds to nucleotides 479-486, of SEQ. ID NO. 2, and whereinthe age-related disease is selected from the group consisting of:neurodegenerative disease, cancer, type 2 diabetes, hypertension, oculardisorders, cataracts, glaucoma, osteoporosis, blood clotting disorders,arthritis, hearing loss, stroke and Alzheimer's disease.
 2. The methodaccording to claim 1 wherein the SIRT6 expression inhibitor comprisesmicro-RNA.
 3. The method according to claim 2 wherein the SIRT6expression inhibitor comprises one or more of SEQ. ID NO. 3, SEQ. ID NO.4.
 4. The method according to claim 1 wherein the agent comprises one ora combination of one or more of SEQ. ID NO. 7, SEQ. ID NO.
 8. 5. Themethod according to claim 1 wherein the agent is in the form of acomposition in combination with a pharmaceutically acceptable carrier.6. The method according to claim 5 wherein the composition isadministered parenterally.
 7. The method according to claim 5 whereinthe composition is administered intravenously.
 8. The method accordingto claim 1 wherein the subject is aged above 65 years old.
 9. The methodaccording to claim 1 wherein the subject is aged above 80 years old. 10.The method according to claim 1 wherein the daily dosage of agentadministered is between 0.01 and 3000 mg daily.
 11. The method accordingto claim 1 wherein the age-related disease is selected from the groupconsisting of ocular disorders, cataracts and glaucoma.
 12. A method fortreating an age-related disease comprising administering to a humansubject above the age of 50 in need thereof an effective amount of anagent which increases the activity of sirtuin-6 (SIRT6) by increasingthe expression of the SIRT6 gene, wherein the agent inhibits binding ofa SIRT6 expression inhibitor to a 3′UTR region of human mRNA encodingSIRT6, wherein the expression inhibitor binds to nucleotides 386-394,nucleotides 199-206, nucleotides 350-359 or nucleotides 455-463 of SEQ.ID NO. 2, and wherein the age-related disease is selected from the groupconsisting of: neurodegenerative disease, cancer, type 2 diabetes,hypertension, ocular disorders, cataracts, glaucoma, osteoporosis, bloodclotting disorders, arthritis, hearing loss, stroke and Alzheimer'sdisease.
 13. The method according to claim 12 wherein the SIRT6expression inhibitor comprises micro-RNA.
 14. The method according toclaim 13 wherein the SIRT6 expression inhibitor comprises one or more ofSEQ. ID NO. 5 and SEQ. ID NO.
 6. 15. The method according to claim 12wherein the agent comprises one or a combination of one or more of SEQ.ID NO. 9 and SEQ. ID NO.
 10. 16. The method according to claim 12wherein the agent is in the form of a composition in combination with apharmaceutically acceptable carrier.
 17. The method according to claim16 wherein the composition is administered parenterally.
 18. The methodaccording to claim 16 wherein the composition is administeredintravenously.
 19. The method according to claim 12 wherein the subjectis aged above 65 years old.
 20. The method according to claim 12 whereinthe subject is aged above 80 years old.
 21. The method according toclaim 12 wherein the daily dosage of agent administered is between 0.01and 3000 mg daily.
 22. The method according to claim 12 wherein theage-related disease is selected from the group consisting of oculardisorders, cataracts and glaucoma.